Technical Field
The present invention relates to the field of plant molecular biology and plant genetic engineering. Specifically, the present invention relates to a glyphosate-resistant/tolerant gene of plant origin and a protein encoded thereby, to a method for obtaining a highly glyphosate-resistant gene mutant by genetic engineering by artificial mutation, and to a highly glyphosate-resistant/tolerant gene of plant origin obtained after artificial mutation. The gene is expressed in a plant by genetic transformation to allow the plant to be glyphosate-resistant, thereby selectively controlling the weed in the crop fields by using glyphosate. The present invention is also applicable to crop breeding, and screening of plant cell culture.
Related Art
Since the initial use of 2,4-D in 1946, chemical herbicides has had a history of over 60 years, and made an enormous contribution to the global food production and agricultural modernization (Powels and Yu, 2010). Among them, glyphosate (N-(phosphonomethyl)glycine) is the most important and widely used herbicide by far (Duke and Powles, 2008). Since its development in 1974 by the Monsanto Company, glyphosate quickly occupied a leading position in the world's herbicide market because of its broad spectrum, low toxicity, safety, no residue in soil and other characteristics. The mechanism of toxic action of glyphosate is mainly by competitively inhibiting EPSPase, that is, 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), which is widely present in fungi, bacteria, algae, and higher plants, but not in animals. Glyphosate is an analogue to and has a molecular formula closely similar to that of phosphoenolpyruvic acid (PEP). Glyphosate can compete with PEP for binding to the EPSPase to form a EPSPase • 3-shikimate-3-phosphate (S3P)• glyphosate complex, whereby the synthesis of EPSP and thus the synthesis of aromatic amino acids are blocked, leading to the death of plants. The non-selectivity in killing the crops and weeds limits the use of glyphosate in, and causes great loss to agricultural production. Glyphosate is widely used in the control of weeds in crop fields only after glyphosate-resistant transgenic crops (e.g. soybean, corn, cotton and rape) are developed and commercialized in 1996 (Powles, 2008). At present, glyphosate is the pesticide with the largest production that has a market share of nearly 20% in the global pesticide market, and has an annual sale of more than 2,000,000,000 $. New glyphosate-resistant crop varieties are cultivated by many researchers through various methods.
At present, it is the most effective means to obtain resistant crops by breeding glyphosate-resistant plants by genetic engineering. In the genetic engineering methodologies, a resistant EPSPase encoding gene of bacterial origin is generally utilized, the product of which cannot be competitively inhibited by glyphosate due to a decreased binding activity, thus ensuring the normal synthesis of aromatic amino acids in plants. The commonly used resistant genes may be derived from Salmonella typhimurium, Agrobacierium tumefaciens, Escherichia coli, Pseudomonas, and so on. Transgenic plants expressing bacterial EPSPase and exhibiting glyphosate resistance are obtained by many researches through genetic engineering methodologies using the resistant genes (Sust and Amrherin, 1990; Blackburn and Boutin, 2003; Maskell, 1998; Gallo and Irvine, 1996; Zhou et al., 1995; Mannerlof et al., 1997; Penaloza et al., 1995; Zboinskaetal., 1992). Among them, glyphosate-resistant transgenic soybean is massively planted in the United States, Brazil and other countries. However, as food or a food material, the safety of the crops with the resistant genes of bacterial origin is persistently a focus of argument.
It is also found in plants that mutation of a single amino acid site in EPSPase results in the resistance to glyphosate. Eleusine indica has an 8 to 12 fold increased resistance to glyphosate, due to the mutation of proline at position 106 in EPSPS to serine or threonine (Pro106Ser/Thr) (Baerson et al., 2002; Ng et al., 2003). Different varieties of glyphosate-resistant Lolium rigidum have 2 different mutations of proline at position 106 in EPSPS, that is, substitution with threonine or alanine (Pro106Thr/Ala) (Wakelin and Preson, 2006; Yu et al., 2007). Mutation of the amino acid at position 182 in EPSPase from proline (P) to serine (S) is one of the mechanisms underlying the glyphosate resistance in Lolium multiflorum (Gonzalez-Torralva, 2012). The affinity to glyphosate is reduced through gene mutations at these effective sites, thereby enhancing the resistance of the weeds to glyphosate.
Furthermore, it is confirmed through studies on glyphosate-resistant EPSPS genes in weeds that the resistance of weeds to glyphosate is largely caused by the change in polarity resulting from amino acid mutations. The non-polar proline at position 106 in EPSPS encoding gene in glyphosate-resistant Eleusine indica and Lolium rigidum is mutated respectively to polar serine and threonine (Baerson, 2002; Wakelin and Preston, 2006). The amino acids at position 101 in EPSPS gene of glyphosate-resistant Lolium rigidum and Convolvulus arvensis L are mutated respectively from non-polar proline and non-polar phenylalanine to polar serine (Simarmata and Penner, 2008; Zhang et al., 2011). The change in the polarity of amino acids may affect the affinity of EPSPase to glyphosate.
A patent is filed for the glyphosate-resistant EPSPS gene of Eleusine indica by the Monsanto Company. However, to improve the resistance level of transgenic crops and increase the diversity of resistant genes, there is still a need in production applications for new glyphosate-resistant/tolerant genes and glyphosate-resistant/tolerant plants based thereon. Compared with the glyphosate-resistant genes of bacterial origin and other heterogeneous genes from other categories, the ecological environmental and food safety risks of the glyphosate-resistant/tolerant crops bred with the evolutionary or natural resistant gene of plant original are lower, whereby the public acceptance is improved.